The enzyme 5-lipoxygenase is a dioxygenase which catalyzes the addition of molecular oxygen to the Delta5 carbon of arachidonic acid. It is the initial enzyme in the metabolic cascade which leads to the production of 5-L-hydroxy,6,8-trans,11,14-cis-eicosatetraenoic acid (5-HETE), 5(S), 12(R)-dihydroxy,6,14-cis-8,10-trans-eicosatetraenoic acid (leukotriene B4 [LTB4] in human granulocytes and to the slow reacting substance of anaphylaxis (SRS-A) leukotrienes C4, D4 and E4 in other cells. The 5-lipoxygenase occupies a role in this cascade analogous to that of cyclooxygenase in the production of the prostaglandins. 5-HETE and LTB4 are chemotactic for human polymorphonuclear leukocytes, and the leukotrienes C4, D4 and E4 have spasmogenic activity equal to or greater than histamine. The physicochemical and kinetic characterization of the 5-lipoxygenase is thus crucial to understanding the control of the generation of these mediators of inflammation. By analogy to the soybean lipoxygenase and bovine seminal vesicle cyclooxygenase, it is possible that the action of the 5-lipoxygenase might be controlled by a kinetic mechanism whereby the enzyme undergoes a self-catalyzed destruction, is both activated and inhibited by its hydroperoxy product, and is inhibited by both its hydroxylated derivatives and by the leukotrienes C4, D4, and E4. The prostaglandins PGD2, PGE2, PGF2Alphs, PGA1, PGA2, PGB1 and PGB2 might act as inhibitors via their hydroxyl groups. The subcellular localization of the 5-lipoxygenase of human polymorphonuclear leukocytes will be determined, and the cofactor requirements examined. The enzyme will then be purified using ion exchange and gel filtration chromatography and isoelectric focusing. Affinity chromatography will also be employed. The reaction products will be identified by both high performance liquid chromatography, thin layer chromatography and conventional spectrophotometric techniques. When the enzyme is purified we will then study its mechanism of control. The long-term goals os this project are to utilize our understanding of the structural and kinetic properties of the enzyme to develop clinically useful specific inhibitors.